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New roles for the cytoskeleton in cell-autonomous immunity to mycobacteria

Periodic Reporting for period 1 - MYCO TRAPS (New roles for the cytoskeleton in cell-autonomous immunity to mycobacteria)

Reporting period: 2016-07-01 to 2018-06-30

Cell-autonomous immunity is the ability of a host cell to eliminate an invasive infectious agent. Recent work has shown that components of the cytoskeleton have a major role in cell-autonomous immunity and control of bacterial infection. The Mostowy group has shown that some intracellular bacteria, including Mycobacterium marinum and Shigella spp, can invade the host cell cytosol where they can interact with cytoskeleton components to form actin tails or be entrapped in septin cage-like structures. However, our knowledge of these interactions derives from a limited number of in vitro studies, and have not been fully characterized in vivo using animal models. Using high-resolution microscopy techniques and state-of-the-art genome editing tools, we proposed to study M. marinum and Shigella interactions with the cytoskeleton, and investigate the role of these interactions in cell-autonomous immunity using infection of tissue culture cells (in vitro) and zebrafish (in vivo). This may provide vital clues towards new strategies aimed at combating infectious diseases, and possibly other human diseases that arise from dysfunctional host responses.

During the fellowship the fellow also addressed the pathogenesis Shigella sonnei, an emerging global threat displacing a niche historically occupied by S. flexneri as the leading etiological cause of bacillary dysentery. Using the zebrafish infection model, it was found that S. sonnei is significantly more virulent than S. flexneri in vivo. The fellow identified the virulence factor responsible for increased virulence, and the mechanism by which this mediates immune evasion.
In the project proposal, the researcher proposed to pursue the following objectives:

Objective 1: To identify and characterize host and pathogen factors controlling autophagy-cytoskeleton interactions and inflammation.
- Over recent years, the lab and others have shown in vitro that M. marinum and S. flexneri lead to rearrangements of the actin and septin cytoskeletons, forming actin tails and septin cages respectively. In particular, we showed that septin cages target bacteria to autophagy both in vitro and in vivo (Mostowy et al, Cell Host Microbe, 2010; Mostowy et al, Plos Pathog, 2013; Sirianni et al, EMBO Rep 2016; Torraca et al, Front Cell Dev Biol 2016, Torraca et al, Trends Cell Biol 2018).
- The fellowship has significantly contributed to the discovery of a previously unknown mechanism by which septins control activation of the inflammasome, and prevent detrimental inflammation during infection (Mazon-Moya et al, Plos Pathog 2017).

Objective 2: To investigate the discovered molecules and mechanisms in vivo using a M. marinum or Shigella-zebrafish infection model.
- Recruitment of septins was observed in zebrafish around S. flexneri in a tailfin and intramuscular infection model. In similar conditions, recruitment of septins around mycobacteria was also observed, but is limited to <5% of bacteria. Recruitment of autophagy to mycobacteria was also observed (limited to ~20% of bacteria), but poor co-localization of Mycobacterium-containing autophagosomes and septins was observed using tools currently available.
- Rearrangement of the actin cytoskeleton was monitored during early and late stages of mycobacterial infection. Phalloidin staining of fixed samples was initially used but failed to capture actin tail formation. However, the fellow generated a transgenic zebrafish line labelling the actin cytoskeleton in macrophages. Recruitment of actin to phagocytic cups and around Mycobacterium-containing phagosomes were observed, but actin tail formation was not clearly observed in vivo.
- The lab also generated a transgenic line to follow Septin 6 in macrophages (mpeg1:mcherry-Sept6) and a knock-in line with precise introduction of monomeric GFP in fusion at the Septin15 locus (Sept15:mGFP-Sept15).
- The fellowship contributed to the development of 5 septin gene KO zebrafish lines (at least one for each septin subgroup) namely Septin 2, Septin 6, Septin 7a, Septin 9a and Septin 15. All lines are viable and do not have any obvious phenotype in the heterozygote carrier stage.
- Using our Shigella-zebrafish infection model, the fellow contributed to the discovery of a novel mechanism by which emergency granulopoiesis induced upon sublethal bacterial infections can boost innate immune defence (Willis et al. mBio, 2018).
- The fellow also worked in collaboration with the Sanger Institute (Cambridge, UK) in a RNA-seq based screen for candidate genes conferring susceptibility/resistance to different bacteria, including Shigella. The screen successfully identified several candidates for downstream analysis.
- The fellow has extended the zebrafish-S. flexneri infection model to S. sonnei, an emerging global threat displacing a niche historically occupied by S. flexneri as the leading etiological cause of bacillary dysentery. Strikingly, S. sonnei is significantly more virulent than S. flexneri in vivo. The fellow identified the precise virulence factor responsible for increased virulence, and the mechanism by which this mediates immune evasion (Torraca et al, mns in prep).

Results obtained from this project will be further explored in the next 2 years by the fellow and the supervisor in their new research environment at the LSHTM.
- To continue research on septins and cell-autonomous immunity, the mutants and the transgenic lines obtained during this fellowship will be used to follow septin rearrangement during infection and precisely dissect the contribution of septins to counteract invading pathogens.
- To complete their research on S. sonnei pathogenesis, the fellow and the supervisor will generate results using human neutrophils to validate their findings from zebrafish. They are additionally setting up collaboration to obtain a cohort of clinical data to corroborate the relevance of their findings in humans. The lab is also generating a dual-host pathogen transcriptome dataset profiling S. sonnei infected zebrafish larvae. It is expected this will significantly contribute to future research avenues studying S. sonnei.
Overall the fellowship contributed to elucidate the implication of the cytoskeleton in cell-autonomous immunity and in the control of bacterial infections. It also contributed to the discovery of a new mechanism by which emergency granulopoiesis can be exploited to boost innate immune defence. The host genes and pathways studied in the project may represent novel targets for host-directed treatment strategies against Shigella and other infectious/inflammatory diseases.

The project has also served to significantly strengthen the researcher’s knowledge and experience in infection, immunity, host-pathogen interactions, inflammation, autophagy, cytoskeleton dynamics, and cellular microbiology. It also broadened his expertise in zebrafish infection models, and may directly translate into a better understanding of human infection. Throughout the project the fellow has acquired highly valuable new skills and collaborations that will be instrumental in his future career, as the researcher aims to develop his own research avenue in the field of inflammation and infectious diseases. The fellow has significantly strengthened his intentions of pursuing an academic career and has recently obtained an additional 2-year postdoctoral position at LSHTM to complete his work and to develop an independent career plan.